Method for characterizing particles in liquid using a dual sample cartridge

ABSTRACT

A method for characterizing particles suspended in a liquid, in a self-contained disposable cartridge for single-use analysis, such as for single-use analysis of a small quantity of whole blood. The method characterizes particles in liquid and samples a small and accurate volume of liquid. The cartridge includes a housing having a mixing chamber and a collection chamber separated by a wall containing an opening, a first bore in the outer surface of the housing for entrance of a liquid sample, a first cavity for receiving and holding a first liquid sample, and a second cavity for receiving and holding a second liquid sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of application Ser. No.11/815,884, now U.S. Pat. No. 8,028,566, filed Sep. 13, 2007, which isthe national phase under 35 U.S.C. 371 of PCT International ApplicationNo. PCT/DK2006/000080 which has an international filing date of Feb. 10,2006, and also claims priority under 35 U.S.C. 119 to Danish applicationPA 2005 00199 filed on Feb. 10, 2005, and U.S. provisional application60/655,416 filed on Feb. 24, 2005, all of which applications are herebyincorporated by reference in their entirety for all purposes as if fullyset forth herein.

BACKGROUND OF THE INVENTION

The present invention relates to an apparatus for characterizingparticles suspended in a liquid, especially a self-contained disposablecartridge for single-use analysis, such as for single-use analysis of asmall quantity of whole blood.

DESCRIPTION OF THE BACKGROUND ART

Present instruments for particle characterization such as counting andsizing are fairly expensive, immobile and require operation by trainedpersonnel. The consequence hereof has been that many instruments areplaced in dedicated laboratories that are operated by specializedpersonnel. Furthermore, the samples to be analyzed must be transportedto this laboratory and the results are reported back to the requiree.

However, efforts have been made to provide a disposable cartridge forparticle characterization. WO 03/104772 discloses a cartridge foranalysis of a blood sample. The cartridge disclosed in WO 03/104772 canbe used for determination of the content of haemoglobin and for countingand differentiation between three types of white blood cells (WBCs). Inone embodiment the platelets are counted after lysing of the bloodsample. In another embodiment the cartridge comprises two orifices andtwo mixing chambers for characterization of a blood sample. The bloodsample is diluted in a first mixing chamber for particlecharacterization of WBCs through a first orifice and a part of thediluted sample is further diluted in a second mixing chamber forparticle characterization of red blood cells (RBCs) and platelets (PLTs)through a second orifice.

WO 03/044488 discloses a disposable apparatus for use in blood testing,the apparatus being adapted for simultaneous dilution of blood into twodifferent dilution ratios in two different mixing chambers.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an apparatus for amore accurate characterization of particles, e.g. platelets, RBCs andWBCs and their subpopulations, such as granulocytes, monocytes, andlymphocytes, in a liquid, such as blood.

It is another object of the present invention to provide an apparatusthat has a simple flow system for counting of platelets and RBCs.

According to the present invention, the above-mentioned and otherobjects are fulfilled by an apparatus for characterizing particles inliquid, comprising a housing with a mixing chamber and a collectionchamber separated by a wall containing an opening for the passage of theparticles between the mixing chamber and the collection chamber. Thehousing may further comprise particle characterization means forcharacterizing particles passing through the opening, a first bore inthe outer surface of the housing for entrance of liquid and a firstcavity for receiving and holding a first liquid sample, the first cavitybeing movably positioned in relation to the housing in such a way that,in a first position, the first cavity is positioned for entrance of thefirst liquid sample into the first cavity, and, in a second position,the first cavity is in communication with the mixing chamber fordischarge of the first liquid sample into the mixing chamber. Thehousing may further comprise a second cavity for receiving and holding asecond liquid sample, the second cavity being movably positioned inrelation to the housing in such a way that, in a first position, thesecond cavity is in communication with the first bore for entrance ofthe second liquid sample into the second cavity, and, in a secondposition, the second cavity is in communication with the mixing chamberfor discharge of the second liquid sample into the mixing chamber.

Preferably, in their first positions, the first cavity, the secondcavity and the first bore are connected in series for entrance of thefirst and second liquid samples into the first cavity and the secondcavity, respectively. In the first positions of the cavities, aconnecting channel may be provided to interconnect the first cavity andthe second cavity.

Alternatively, in the first positions of the cavities, the first cavityand the second cavity may be connected in parallel with the first borefor entrance of the first and second liquid samples into the firstcavity and the second cavity, respectively.

In a preferred embodiment, the first cavity is positioned in a firstsampling member movably positioned in the housing, and the second cavityis positioned in a second sampling member movably positioned in thehousing. In a first position of the second sampling member, i.e. a firstposition of the second cavity, the second cavity is in communicationwith the first bore. Preferably, in the first position of the secondsampling member, the second cavity is in communication with a connectingchannel, which, in the first position of the first sampling member, i.e.the first position of the first cavity, is in communication with thefirst cavity, such that the first bore, the first cavity in the firstposition, the connecting channel, and the second cavity in the firstposition form a channel for entrance of liquid into the first cavity andthe second cavity. In a second position of the first sampling member andthe second sampling member, the first cavity and/or the second cavitymay be in communication with an inlet to the mixing chamber,respectively.

In another embodiment, the first cavity and the second cavity arepositioned in a first sampling member that is movably positioned in thehousing. In this embodiment, in the first position of the first samplingmember, i.e. in the first positions of the first cavity and the secondcavity, the first cavity and the second cavity are in communication witha connecting channel and the first bore in the outer surface of thehousing for entrance of liquid, such that the first bore, the firstcavity, the second cavity, and the connecting channel form a channel forentrance of liquid into the first and second cavity. In a secondposition of the first sampling member, i.e. a second position of thefirst cavity, the first cavity may be in communication with an inlet tothe mixing chamber. In a third position of the first sampling member,i.e. a second position of the second cavity, the second cavity may be incommunication with an inlet to the mixing chamber.

Preferably, the sampling member or members are rotatable about an axisof rotation that is substantially perpendicular to a longitudinal axisof their respective cavities.

Additionally or alternatively, the sampling member or members may bedisplaced in a direction substantially perpendicular to a longitudinalaxis of one of the first and second cavity.

Preferably, the sampling member or members are made of a polymer.

The housing may further comprise a second bore in the outer surface ofthe housing for entrance of liquid. In the first position, the firstcavity may be in communication with the second bore for entrance of thefirst liquid sample into the first cavity.

Thus, the first cavity and the second cavity in the first samplingmember and/or second sampling member receive and hold first and secondsamples of precise volume of liquid, respectively, and the firstsampling member and/or the second sampling member operate to transferthe first sample and the second sample to an inlet of the mixingchamber.

Preferably, liquid to be sampled enters the respective cavities bycapillary attraction causing a liquid flow. Utilization of capillaryforces simplifies the flow system, since no pumps, membranes, syringesor other flow generating means are needed to take the sample.

Thus, the first bore and/or second bore may form capillary tunnel(s) forentrance of liquid by capillary attraction. The capillary tunnel(s)is/are dimensioned so that, upon contact between the bore and liquid tobe sampled, a sample of the liquid is drawn into the bore by capillaryattraction.

Further, the first cavity may form a capillary tunnel adapted fordrawing the liquid sample into the first cavity by capillary attraction,the second cavity may form a capillary tunnel adapted for drawing theliquid sample into the second cavity by capillary attraction, and theconnecting channel may form a capillary tunnel adapted for drawing theliquid sample into the connecting channel by capillary attraction.

The capillary tunnels may together form a capillary tunnel.

Preferably, the first cavity and the second cavity are channels withdifferent diameter, e.g. the second cavity has a larger diameter thanthe first cavity to enhance capillary effect when the first samplingmember and/or the second sampling member are in their first positions.Further, it is preferred that the first cavity and the second cavity intheir first positions extend along substantially the same longitudinalcenter axis.

The first and/or second sampling members may comprise at least onerecess in their surface, such that the at least one recess and anabutting surface of the housing define the first cavity and/or thesecond cavity for receiving and holding first and/or second liquidsamples.

Preferably, the first and second liquid samples have different volumes.Counting of platelets and RBCs usually requires a blood sample to bediluted in the range from about 1:2.000 to about 1:100.000, preferablyabout 1:10.000. Counting of WBCs after lysing of a blood sample usuallyrequires a blood sample to be diluted in the range from about 1:100 toabout 1:2.000, preferably 1:500. The different dilution ratios of thesamples for counting of platelets/RBC and WBC, respectively, may beobtained by adjusting the volume of the sample and/or the volume of theliquid in one or more liquid storage chambers for dilution and furthertreatment of blood samples.

The surfaces of the inner capillary tunnel walls may be hydrophilicwhereby the capillary attraction of the liquid sample is facilitated.For example, the inner tunnel walls may be made of e.g. glass orpolymers, such as polystyrene.

Alternatively, the capillary tunnel walls may be made of another type ofmaterial and covalently or non-covalently coated with a hydrophilicmaterial, such as a polymer or a reagent.

The first and/or second cavities may have an anti-coagulation reagent onits surface. Further, the capillary tunnels may also include one or morereagents adhered or chemically bonded to the inner tunnel walls. Thesereagents serve the purposes of further facilitating the capillaryattraction of the sample and optionally also causing a chemical reactionin the liquid sample, e.g. introducing anticoagulant activity in a bloodsample. Such reagents may comprise heparin, salts of EDTA, etc.

The volume of the first cavity and thus substantially the volume of thefirst sample may range from 0 to 10 μL, such as from 0.1 μL to 1 μL,preferably about 0.2 μL. The volume of the second cavity and thussubstantially the volume of the second sample may range from 0 to 100μL, such as from 0.5 μL to 10 μL, preferably about 2 μL.

The particle characterization means may include a first electrode in themixing chamber and a second electrode in the collection chamber, eachelectrode being electrically connected to a respective terminal memberaccessible at the outer surface of the housing.

The housing may further comprise a first liquid storage chamber forholding liquid for first and/or second sample treatment. In the secondposition of the first cavity, the first cavity may be in communicationwith the first liquid storage chamber for flushing the first sample intothe mixing chamber.

Additionally, the housing may comprise a second liquid storage chamberfor holding liquid for first and/or second sample treatment. In thesecond position of the second cavity, the second cavity may be incommunication with the first liquid storage chamber and/or the secondliquid storage chamber for flushing the second sample into the mixingchamber.

Preferably, the first liquid storage chamber and the second liquidstorage chamber are constructed to facilitate total draining of thechambers.

Preferably, the first liquid storage chamber and/or the second liquidstorage chamber contain a diluent, e.g. water, or other liquids such asreagents, solvents, lysing agents or suitable solutions for sampletreatment.

Preferably, the housing of the apparatus constitutes a cartridge for asingle analysis of a dual sample blood portion.

In accordance with a further aspect of the invention, an apparatus isprovided for characterizing particles suspended in a liquid, comprisinga housing as disclosed herein constituting a cartridge, and a dockingstation for removably receiving the cartridge, the docking stationcomprising connectors for operational connection with the particlecharacterization means when the cartridge is received in the dockingstation.

The housing, e.g. constituting a cartridge, may further comprise a firstport communicating with the mixing chamber for causing a liquid flowfrom the liquid storage chambers through the first cavity and the secondcavity to the mixing chamber, and the docking station may furthercomprise a corresponding port for forming a gas connection with thefirst port when the cartridge is received in the docking station forapplication of a pressure causing a liquid flow through the first cavityand the second cavity. In one embodiment of the present invention,application of a pressure on the first port of the housing may cause aliquid flow through the opening.

Preferably, the housing further comprises a second port communicatingwith the collection chamber for causing a liquid flow through theopening, and the docking station may further comprise a correspondingport for forming a gas connection with the cartridge port when thehousing is received in the docking station for application of a pressurecausing a liquid flow through the opening. In one embodiment of thepresent invention, liquid flow through the opening may be obtained byapplication of a pressure on the first port of the housing.

One or more pistons or membranes may be integrated into the housing toinclude a source of pressure for causing a liquid flow in the housing.The docking station may provide a mechanical force for moving the one ormore pistons or membranes. Furthermore, the docking station may beadapted to move, e.g. around an axis by rotating and/or along an axis bypushing and/or pulling, the first sampling member and/or the secondsampling member into different positions, e.g. first positions, secondpositions and/or third positions.

The particle characterization means may include a first electrode in themixing chamber and a second electrode in the collection chamber, eachelectrode being electrically connected to a respective terminal memberaccessible at the outer surface of the housing for operationalconnection to the respective connector of the docking station when thehousing is received in the docking station.

Generally, it is preferred that all necessary mechanical, electrical andfluid connections to the housing can be established by fitting thehousing constituting a cartridge into the docking station, preferably bya simple push fit for simple insertion and removal of the housing.

The first and second electrodes may facilitate particle characterizationutilizing the well-known Coulter impedance principle, e.g. for countingand sizing of blood cells. This method has become a globally acceptedmethod and is being used in the majority of haematology-analysers.Several thousand particles per second may be characterized with highprecision and accuracy utilizing this principle.

The opening in the wall between the mixing chamber and the collectionchamber may be in the form of an orifice, a channel or a duct.Preferably, the opening is an orifice.

With the electrical impedance technique it is possible to resolve theparticle volume from the measurement. By maintaining a constant currentacross the opening or orifice, the recorded voltage pulse from particlesdisplacing the electrolyte in the orifice will have a heightproportional to the volume of the particle. This is because particlescan be considered non-conducting compared to the electrolyte, theelectrical field (DC or RF) in the centre of the orifice is homogeneous,which is normally the case when the diameter D is smaller than thelength l of the orifice (l/D>1), the particle d is to be consideredsmall compared to the diameter of the orifice (d<0.2*D), only oneparticle passes through at a time, and the particles are passed throughthe orifice along the length of the orifice.

Normally such apparatus is operated so that the flow through the openingis into the collection chamber.

Preferably, the length of the orifice is from 1 μm to 1000 μm, forexample about 50 μm. Desirably the length of the orifice is chosen suchthat only one particle will be present in the orifice at the time whendetecting particles of from 0.1 μm to 100 μm diameter. However,considerations to the homogeneity of the electrical field in the orificemay require a length of the orifice larger or equal to the diameter. Thecounts, of which some may be simultaneous counting of two particles, canbe corrected mathematically by implementing a statistical estimation.The aspect ratio of the orifice, (length or depth divided by diameter)is preferably from 0.5:1 to 5:1, more preferably from 1:1 to 3:1.

Preferably, the largest cross-sectional dimension of the orifice is from5 μm to 200 μm, for example 10 μm to 50 μm.

Preferably, the wall between the mixing chamber and the collectionchamber comprises a membrane with an orifice for passage of particlesand/or liquid between the mixing chamber and the collection chamber.

As explained above, the present invention provides in preferred aspectsa sensor based on a membrane fabricated in e.g. a polymer sheet. Orificeformation with high precision and high reproducibility can be fabricatedby laser ablation. The membrane has an orifice placed relatively in thecentre of the membrane, which can be used for aspiration of particlessuspended in a liquid, as the sensor is submerged into the liquid. Thisway of transporting particles into a measuring region is known forelectrical characterization of particles by the Coulter principle (V.Kachel, “Electrical Resistance Pulse Sizing: Coulter Sizing”, FlowCytometry and Sorting, 2. ed., pp 45-80, 1990 Wiley-Liss, Inc.).

The housing may further comprise one or more breather inlet/outletscommunicating with the surroundings for preservation of substantiallyambient atmospheric pressure in the housing flow system for facilitationof liquid flow in the housing flow system e.g. through the opening. Oneor more breakable seals may be provided for sealing one or more breatherinlet/outlets during transport and storage. A breather inlet/outlet mayconnect a chamber and the surroundings for preservation of substantiallyambient atmospheric pressure in the chamber.

Preferably, the housing constitutes a cartridge that is designed to bedisposable after a single use. It is desirable that after use there isno need to clean the apparatus before it can be used in a new assayprocedure with a new cartridge. Accordingly, escape of liquid from thecartridge at its entry into the docking station should be avoided.Preferably, a volume of liquid sufficient for the desired particlecharacterization can be drawn or pumped through the opening without theliquid passing out of the housing. Generally, it should be possible topass a total volume of liquid, which is at least 0.1 ml to 10 ml, e.g.1.5 ml, through the opening whilst particle characterizationmeasurements are being made with no liquid leaving the housing.

The housing may comprise volume-metering means for determining thebeginning and end of one or more, e.g. two or three, periods duringwhich a predetermined volume of liquid has passed through the opening.Preferably, the one or more periods comprise a period for countingplatelets and RBCs and a period for counting and differentiating betweenWBCs.

Preferably, the volume-metering means comprises a first volume-meteringchamber with an input, e.g. communicating with the collection chamber,and an output, and wherein presence of liquid is detected at the inputand at the output, respectively.

Further, the volume-metering means may comprise a second volume-meteringchamber with an input communicating with the output from the firstvolume-metering chamber and an output, and wherein presence of liquid isdetected at the input and at the output, respectively.

Preferably, the volume-metering means comprise one or more detectionmeans. Preferably, the one or more detection means are positioned forfacilitating sensing or determining, when liquid in the metering meansis at or above respective levels in the volume-metering means, forexample when a volume-metering chamber is filled with liquid.Preferably, the inputs and outputs of respective volume-metering chamberor chambers are provided with detection means.

The detection means may be optical detection means or optical detectionparts, i.e. presence of liquid may be detected optically due to changedoptical properties of an optical detection part, e.g. a channelconfiguration, from being filled with air till when it is being filledwith liquid. This could be constructed as reflectance or transmittancedetection from the surface, where incident light is reflected from anempty channel and transmitted through a filled channel, thus giving aclear shift in the detected reflected or transmitted light.Alternatively or in combination with the optical detection means, thedetection means may comprise electrical sensors.

It is preferred that the inputs and the outputs of the metering chambersare formed by narrow channels for accommodation of only a small liquidvolume compared to the volume of the metering chambers so that theactual positioning of the detection means, e.g. optical reflectancedetection, in the channels do not substantially influence the accuracyof the volume metering determination.

The mixing chamber and/or the collection chamber may constitute one ofthe volume-metering chambers; however, it is preferred to provideindependent volume-metering chambers facilitating positioning of thedetection means, e.g. detection means for optical reflectance detection.

The volume-metering means may be used for sensing when the level of theliquid is such that a respective metering chamber or chambers are empty,filled, partly filled or not filled with liquid and may therefore servefor determining the beginning and/or end of one, two, three, or moreperiods during which a fixed volume of liquid has passed through theopening. For example, a first period of particle characterization, e.g.counting of platelets and RBCs, may begin when the level of the liquidjust reaches or rises over the level of a first detection means and mayend when the level of the liquid just reaches or rises over a seconddetection means, the volume of liquid passing through the opening duringthis period being defined by the volume of the space between therespective detection means. Further a third and/or a fourth detectionmeans may be provided for determining the beginning and/or end of asecond period of particle characterization, e.g. counting anddifferentiation of WBCs.

The housing may further comprise an overflow chamber for accommodationof liquid after passage through the opening.

A mixing member may be positioned in the mixing chamber. The mixingmember may be a magnetic mixing member.

A part of the housing, e.g. the mixing chamber, may be adapted forspectrophotometric characterization, e.g. determination of haemoglobinin a liquid sample. The mixing chamber or other parts of the housing maycomprise one or more windows to facilitate the spectrophotometriccharacterization.

The housing may further comprise a pump chamber communicating with thecollection chamber and may have a pump actuator for causing a liquidflow through the opening. The pump actuator may be a piston or amembrane.

The docking station may comprise a pump device comprising one or morepumps and one or more directional valves for application of a pressureon the first port of the docking station and the second port of thedocking station. Further, the docking station may comprise one or moreengagement members for engagement with the first sampling member and/orthe second sampling member when the cartridge is removably inserted intothe docking station. The pump device and the one or more engagementmembers may be controlled according to a desired measuring cycle.

The docking station may further comprise one or more detectors and/orsensors, e.g. one or more optical detectors, for detecting presence ofliquid in certain parts of the cartridge, e.g. in the detection means ofthe cartridge.

Thus, the apparatus according to the invention provides dual sampleanalysis by the Coulter Principle through one opening, facilitatingdetermination of content of platelets, RBCs and WBCs in blood.

In accordance with a further aspect of the invention a method forcharacterizing particles in liquid is provided, the method comprisingthe steps of:

-   -   a) entering a first and a second liquid sample containing        particles into a first and second cavity, respectively,    -   b) moving a first liquid through the first cavity and into a        mixing chamber together with the first liquid sample,    -   c) performing first particle characterizing measurements by        passage of at least a part of the first liquid sample from the        mixing chamber through an opening and into a collection chamber,    -   d) moving a second liquid through the second cavity and into the        mixing chamber together with the second liquid sample, and    -   e) performing second particle characterizing measurements by        passage of at least a part of the second liquid from the mixing        chamber through the opening and into the collection chamber.

Preferably, the method for characterizing particles in liquid isperformed with an apparatus according to the description above.

The method is particularly intended for analysis of blood.

Preferably, step c) of performing first particle characterizingmeasurements comprises the step of counting of RBCs and platelets byCoulter Counting in at least a part of the first liquid sample.

Preferably, step e) of performing second particle characterizingmeasurements comprises the step of counting and differentiation of oneor more different WBCs in at least a part of the second liquid sample.

Particle characterization performed in steps c) and/or e) may be startedand/or stopped when a prescribed volume of liquid has passed through theopening.

Preferably, step b) and/or step d) comprise moving a mixing member inthe mixing chamber for enhanced mixing of samples and liquid, and stepb) may further comprise the step of priming and/or calibrating theapparatus with a part of the liquid in the mixing chamber.

The mixing ratio between the first liquid sample and liquid, e.g.substantially first liquid, in the mixing chamber just before firstparticle characterization measurements are performed may be in the rangefrom about 1:2.000 to about 1:10.000, preferably about 1:10.000, and themixing ratio between the second liquid sample and liquid, e.g.substantially a mixture of first and second liquids, in the mixingchamber just before second particle characterization measurements areperformed may be in the range from about 1:100 to about 1:2.000,preferably about 1:500.

Preferably, the first liquid is a conductive liquid facilitating Coulteranalysis of the first and second liquid samples.

Preferably, the second liquid is a lysing agent for lysing of RBCs inthe mixing chamber facilitating counting and determination of differentWBCs in the second liquid sample.

Additionally, the content of haemoglobin may be determined byspectrophotometric characterization. Preferably, spectrophotometriccharacterization is performed through a window in the mixing chamberbetween step d) and step e).

According to the present invention, a device for sampling a small andaccurate volume of liquid is provided, comprising a housing comprising afirst connecting part and a second connecting part, and a first samplingmember that is movably positioned in the housing and having a recess inits surface, the recess and an abutting surface of the housing defininga cavity for receiving and holding a liquid sample. In a first positionof the first sampling member, the cavity is in communication with thefirst connecting part and the second connecting part, the firstconnecting part functioning as an inlet for the liquid sample and thesecond connecting part functioning as an outlet for the liquid sample,and, in a second position, the cavity is enclosed within the housing andthe sampling member.

The first connecting part may form a first capillary tunnel and beadapted so that, upon contact between the first connecting part andliquid to be sampled, a sample of the liquid is drawn into the firstconnecting part by capillary attraction.

The cavity may form a capillary tunnel that is adapted for drawing theliquid sample into the cavity by capillary attraction.

The sampling member may be rotatable about an axis of rotation that issubstantially perpendicular to a longitudinal axis of the cavity.

The sampling member may be displaced in a direction substantiallyperpendicular to a longitudinal axis of the cavity.

The housing may further comprise a liquid storage chamber such that theliquid sample in a second position of the cavity is in communicationwith a liquid in the liquid storage chamber.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further described and illustrated in furtherdetail with reference to the accompanying drawings in which:

FIG. 1 schematically shows one embodiment of an apparatus according tothe invention,

FIG. 2 schematically shows the embodiment of FIG. 1 with first andsecond cavities in their second positions,

FIG. 3 schematically shows another embodiment of an apparatus accordingto the invention,

FIG. 4 schematically shows another embodiment of an apparatus accordingto the invention,

FIG. 5 shows a section of the embodiment in FIG. 4 with the first cavityin a second position,

FIG. 6 shows a section of the embodiment in FIG. 4 with the secondcavity in a second position,

FIG. 7 schematically shows yet another embodiment of an apparatusaccording to the invention,

FIG. 8 schematically shows still another embodiment of an apparatusaccording to the invention,

FIG. 9 shows a new sampling principle for sampling very small amounts ofliquid,

FIG. 10 schematically shows yet another embodiment of an apparatusaccording to the invention,

FIG. 11 shows the embodiment in FIG. 10 with the first cavity in asecond position,

FIG. 12 shows the embodiment in FIG. 10 with the second cavity in asecond position,

FIG. 13 schematically shows still another embodiment of an apparatusaccording to the invention,

FIG. 14 schematically shows an embodiment of an apparatus according tothe invention, and

FIG. 15 illustrates one embodiment of the method according to theinvention.

The same reference number denotes corresponding elements in thedifferent embodiments in the figures.

DETAILED DESCRIPTION

FIG. 1 schematically shows one embodiment of the apparatus according tothe invention. The figure shows a disposable cartridge 100 for CompleteBlood Counts (CBC), i.e. counting of red blood cells (RBCs), bloodplatelets (PLTs), white blood cells (WBCs) including a differentialcount of subpopulations of WBCs. Furthermore, the cartridge includesmeans for determination of other blood constituents such as haemoglobin,blood gasses (such as pH) or proteins (such as C-reactive protein) orthe like.

The cartridge 100 comprises a housing 2 with a mixing chamber 4 and acollection chamber 6 separated by a wall 8 containing an opening 10 forthe passage of particles in liquid between the mixing chamber and thecollection chamber. Particle characterization means 12,14 are providedfor characterizing particles passing through the opening 10.

The housing further comprises a first bore 16 in the outer surface ofthe housing for entrance of liquid and a first cavity 18 for receivingand holding a first liquid sample. In a first position as shown in thefigure, the first cavity 18 is positioned for entrance of the firstliquid sample into the first cavity. The first cavity 18 is movablypositioned in relation to the housing 2 in such a way that, in a secondposition, the first cavity is in communication with the mixing chamber 4for discharge of the first liquid sample into the mixing chamber 4.Further, a second cavity 20 for receiving and holding a second liquidsample is provided. The second cavity 20 is movably positioned inrelation to the housing 2 in such a way that, in a first position, thesecond cavity is in communication with the first bore 16 for entrance ofthe second liquid sample into the second cavity 20. In a secondposition, the second cavity 20 is in communication with the mixingchamber 4 for discharge of the second liquid sample into the mixingchamber 4.

In this embodiment, the first cavity 18 extends as a channel through afirst sampling member 22 that can be rotated around an axisperpendicular to the figure, and the second cavity 20 extends as achannel through a second sampling member 24 that can be rotated aroundan axis perpendicular to the figure. The respective channels in therespective sampling members form the first cavity 18 and the secondcavity 20.

In the first position of the first cavity, the first cavity 18 is incommunication with a connecting channel 26 extending from the firstsampling member to the second sampling member, and in the first positionof the second cavity, the second cavity 20 is in communication with thefirst bore 16 and the connecting channel 26. When in their respectivefirst positions as seen in FIG. 1, the first cavity 18 and the secondcavity 20 form a capillary with the first bore 16 and the connectingchannel 26. The first cavity is in communication with an outlet 28 thatallows air to escape out of the capillary and out of the housingallowing sample entry. By turning the first and second sampling members22, 24, precise volumes of liquid, such as blood, are trapped inside thecavities 18, 20.

Preferably, the volumes of the first cavity and the second cavity aredifferent. The volume of the first cavity and thus substantially thevolume of the first sample may range from 0 to 10 μL, such as from 0.1to 1 μL, preferably 0.2 μL. The volume of the second cavity and thussubstantially the volume of the second sample may range from 0 to 100μL, such as from 0.5 to 10 μL, preferably 2 μL.

FIG. 2 shows the embodiment of FIG. 1 with the first cavity 18 and thesecond cavity 20 in their respective second positions.

In the second position of the first cavity, the first cavity 18 is incommunication with the mixing chamber 4. Further, the first cavity 18 isin communication with a first liquid storage chamber 30 through a shortduct. Preferably, first liquid in the first liquid storage chamber 30 ispumped through the first cavity, thereby filling or flushing the firstliquid sample and the first liquid into the mixing chamber 4 thusforming a precisely diluted and analytically prepared blood sample.

In the second position of the second cavity, the second cavity 20 is incommunication with the mixing chamber 4. Further, the second cavity 20is in communication with a second liquid storage chamber 32 through ashort duct. Preferably, second liquid in the second liquid storagechamber 32 is pumped through the second cavity, thereby filling orflushing the second liquid sample and the second liquid into the mixingchamber 4 thus forming a precisely diluted and analytically preparedblood sample.

Blood samples partly or fully constituting first and second liquidsamples in the first cavity and the second cavity, respectively, maythus be diluted and prepared for analysis in the mixing chamber 4. Thesamples are analyzed by impedance sizing, i.e. the Coulter Principle,through the orifice 10 in the wall 8 of the mixing chamber. A conductiveliquid forms an electrical connection from the first electrode 12 in themixing chamber 4 to the second electrode 14 in the collection chamber 6.Changes in impedance of the electrical connection originating from cellspassing in a liquid flow through the orifice 10 can be recorded forcounting and sizing of the cells.

Further, the cartridge comprises a first volume-metering chamber 34 withan input 36 communicating with the collection chamber and an output 38,and wherein presence of liquid is detected at the input and at theoutput, respectively.

Furthermore, the cartridge comprises a second volume-metering chamber 40with an input 42 communicating with the output 38 from the firstvolume-metering chamber 34 and an output 44, and wherein presence ofliquid is detected at the input and at the output, respectively.

The respective inputs and outputs of the metering chambers are formed bynarrow channels for accommodation of only a small liquid volume comparedto the volume of the metering chambers. The narrow channels are a partof the detection means, as the narrow channels are employed for opticalreflectance detection.

The volume-metering chambers 34, 40 can be used to determine and controlvolume for calculating the concentrations of the counted cells and thusdefine periods of measurement.

The housing 2 further comprises a first port 48 communicating with themixing chamber for causing a liquid flow from the liquid storagechambers 30, 32 through the first cavity 18 and the second cavity 20,respectively. A pressure applied to the first port may cause a liquidflow through the first cavity 18 and/or the second cavity 20 into themixing chamber.

A first channel 49 connects the mixing chamber and the first port 48.

The housing 2 further comprises a second port 50 communicating with thecollection chamber for causing a liquid flow through the opening 10. Thesecond port 50 is in communication with the collection chamber via thevolume-metering chambers 34, 40. A pressure applied to the second portmay cause a liquid flow through the opening 10.

A second channel 51 connects the second port 50 and the secondvolume-metering chamber 40.

Spectrophotometric measurements can be established through a smallwindow 46 in the mixing chamber 4.

A method of performing particle characterization according to theinvention is now illustrated by an example of performing CBC in thecartridge in FIGS. 1 and 2. The method for CBC comprises the followingsteps:

-   -   blood is drawn by capillary forces into the first bore 16        filling the first cavity 18 and the second cavity 20 in their        first positions,    -   the first cavity 18 is moved to its second position by turning        the first sample member 22,    -   the first blood sample in the first cavity 18 is diluted a        factor 1:10.000 with an isotonic diluent from the first liquid        storage chamber 30, by pumping first liquid from the first        liquid storage chamber 30 through the first cavity 18 and into        the mixing chamber 4,    -   RBCs and PLTs are counted in a first period for particle        characterization during filling of the first volume-metering        chamber 34 by a liquid flow from the mixing chamber 4 through        the opening 10 to the collection chamber 6 and subsequently to        the first volume-metering chamber 34,    -   the flow through the opening 10 is stopped,    -   the second cavity 20 is moved to its second position by turning        the second sample member 24,    -   the remaining liquid in the mixing chamber 4 is mixed with the        second blood sample from the second cavity 20 and the second        liquid in the second liquid storage chamber 32 to obtain a        substantially 1:500 dilution of the second blood sample, by        pumping second liquid from the second liquid storage chamber 32        through the second cavity 20 and into the mixing chamber 4,    -   the second liquid lyses the RBCs in the remaining first blood        sample (negligible) and the second blood sample and transforms        the haemoglobin into a measurable and stable component. The        concentration of haemoglobin is measured through the window 46        in the mixing chamber 4 by absorption,    -   WBCs are counted in a second period for particle        characterization during filling of the second volume-metering        chamber 40. Counting of WBCs include identification of the WBC        subtypes (three or five part differentials), e.g. lymphocytes,        monocytes and granulocytes.

In one embodiment, first liquid is pumped from the first liquid storagechamber 30 through the first cavity 18 and into the mixing chamber 4 byapplying a first pressure to the collection chamber 4 via the firstchannel 49, wherein the first pressure is higher than the pressure inthe first liquid storage chamber 30. Due to the difference in pressurebetween the first liquid storage chamber 30 and the mixing chamber 4,air bubbles move through the first cavity to substantially equalize thepressure in the first liquid storage chamber 30 and the mixing chamber4. Subsequently, a second pressure is applied to the mixing chamber 4,wherein the second pressure is lower than the pressure in the firstliquid storage chamber 30. Due to the difference in pressure between thefirst liquid storage chamber 30 and the mixing chamber 4, andorientation of the cartridge, at least a part of the first liquid andthe first liquid sample moves through the first cavity into thecollection chamber to substantially equalize the pressure in the firstliquid storage chamber 30 and the mixing chamber 4. This operation maybe repeated until the first liquid storage chamber 30 is substantiallyempty. The first pressure and the second pressure may also be applied tothe collection chamber 6 to substantially avoid liquid transport betweenthe mixing chamber 4 and the collection chamber 6 in this step. Thefirst pressure and/or the second pressure may be applied via the secondchannel 51.

Likewise, second liquid is pumped from the second liquid storage chamber32 through the second cavity 20 and into the mixing chamber 4 byapplying alternating pressures to the mixing chamber 4.

FIG. 3 shows another embodiment of the present invention. The cartridge110 comprises a third volume-metering chamber 52 between the collectionchamber 6 and the first volume-metering chamber 34. Presence of liquidis detected at the input and at the output, respectively. The thirdvolume-metering chamber 52 may be employed to control a third period forcalibration before first and second particle characterization. Further,the housing 2 comprises an overflow chamber 54 in order to preventliquid from flowing out through second port 50. Other embodiments, e.g.embodiments schematically illustrated in FIGS. 1-2 and FIGS. 4-14, mayalso comprise a third volume-metering chamber between the collectionchamber 6 and the first volume-metering chamber 34 and/or an overflowchamber.

Additionally, the mixing chamber 4 may have a small magnetic mixingmember 56 included for forced mixing of liquid by stirring. The mixingmember is rotated by an externally rotating magnetic field that isstrong enough to hold the mixing member oriented according to themagnetic field. Other embodiments, e.g. the embodiments illustrated inFIGS. 1-2 and FIGS. 4-14, may comprise a mixing member.

The presence of liquid in the channels constituting the inputs andoutputs of the volume-metering chambers 34, 40, 52 can be detected byoptical detection means. The refractive index of the interface betweenthe housing and the channel will vary as the channel is filled withliquid instead of air. The incident light from a light source (notshown) will be reflected with an empty channel, and a sensor (not shown)records the reflected light. When the channel is filled with liquid thelight is no longer reflected and the sensor records the change.

FIG. 4 shows another embodiment of the present invention. The cartridge120 comprises a first sampling member 22 comprising the first cavity 18and the second cavity 20. The first sampling member is in a firstposition and thus first and second cavities are in their first positionsfor filling blood into the cavities by capillary forces as describedabove.

FIG. 5 shows a section of the cartridge 120 with the first samplingmember 22 in a second position. The first cavity 18 is in its secondposition and in communication with the mixing chamber 4. Further, thefirst cavity 18 is in communication with a first liquid storage chamber30 through a short duct, thereby connecting the first liquid storagechamber 30 and the mixing chamber 4. The second cavity 20 is not incommunication with the first liquid storage chamber 30

FIG. 6 shows a section of the cartridge 120 with the first samplingmember 22 in a third position. The second cavity 20 is in its secondposition and in communication with the mixing chamber 4. Further, thesecond cavity 20 is in communication with a second liquid storagechamber 32 through a short duct thereby connecting the second liquidstorage chamber 32 and the mixing chamber 4.

FIG. 7 shows another embodiment of the present invention, where thefirst cavity 18 is in its first position and communicates with a secondbore 58 for entrance of liquid to be sampled.

FIG. 8 shows another embodiment of the present invention. In their firstpositions, the first cavity 18 and the second cavity 20 are in parallelcommunication with the first bore 16 for entrance of liquid into thefirst cavity and the second cavity.

FIG. 9 shows a new sampling principle for sampling a very small amountof blood, such as from 0 to 1 μL, e.g. from 0 to 0.5 μL, from about 0.1μL to about 0.3 μL, preferably 0.2 μL. A device for sampling a small anaccurate volume of liquid comprises a housing and a sampling memberhaving a recess in its surface thereby defining a cavity with anabutting surface of the housing. The small cavity 60 in the samplingmember 62 is connected to connecting parts 64, 66, preferablycapillaries, for filling of blood. Preferably, the cavity 60 is a recessin the surface of the sampling member and may contain a very smallamount of blood and thus provide a high dilution rate with reagent inthe liquid storage chamber 68. The small blood sample volume isdifficult to make in a channel, because of the very small diameterrequired. A small cavity in the surface of the sampling member can beused for sampling very small amounts of blood with high reproducibility.

FIG. 9A shows the device with the cavity in the first position beforefilling of blood. FIG. 9B shows the capillaries 64, 66 and cavity 60filled with blood. FIG. 9C shows the sampling member 62 turned into asecond position for diluting the precise amount of blood in the cavity.The cavity is in communication with the liquid storage chamber 68. InFIG. 9D the blood has been diluted with reagent in the liquid storagechamber. Dilution of the blood may take place by stirring or by washingthe cavity with the diluent.

FIG. 10 shows an embodiment 150 of the present invention employing thenew sampling principle for sampling a very small amount of blood asshown in FIG. 9A-D. The first cavity 18 is a recess on the surface ofthe first sampling member 22.

In the first position of the first cavity, the first cavity 18 is incommunication with a connecting channel 26, and in the first position ofthe second cavity, the second cavity 20 is in communication with thefirst bore 16 and the connecting channel 26. The first cavity 18 and thesecond cavity 20 form a capillary with the first bore 16 and theconnecting channel 26.

The collection chamber 6 extends behind the mixing chamber 4, such thata wall containing the opening 10 separates the mixing chamber 4 and thecollection chamber 6 according to the same principle as schematicallyillustrated in FIGS. 1-3. Detection means 70, 72, 74 for detectingpresence of liquid in channels at the inputs and outputs of thevolume-metering chambers 34, 40 are provided for determining and/orcontrolling one, two or more periods of measurement. Priming and/orcalibration of the apparatus may be performed during filling of thecollection chamber, e.g. until the liquid level reaches detection means70.

FIG. 11 shows the embodiment in FIG. 10 with the first cavity 18 in asecond position. The first cavity is in communication with the mixingchamber 4 via a connecting part 76 and a channel 78 in the samplingmember. The first liquid storage chamber 30 is also in communicationwith the first cavity such that first liquid in the first liquid storagechamber and the first liquid sample in the first cavity can be moved tothe mixing chamber 4.

FIG. 12 shows the embodiment in FIG. 10 with the second cavity 20 in asecond position. The second cavity is in communication with the mixingchamber 4. The second liquid storage chamber 32 is also in communicationwith the second cavity such that second liquid in the second liquidstorage chamber and the second liquid sample in the second cavity can bemoved to the mixing chamber 4.

FIG. 13 shows another embodiment of the present invention, where thefirst cavity 18 and the second cavity 20 are comprised in the firstsampling member 22 and connected in parallel with the first bore 16.

FIG. 14 illustrates one embodiment of an apparatus for characterizingparticles suspended in a liquid. The apparatus comprises a housing 150constituting a cartridge 210, and a docking station 220 for removablyreceiving the cartridge 210. It is to be understood that the dockingstation 220 may be adapted for receiving and/or operating otherembodiments of a housing according to the present invention, e.g. theembodiments schematically illustrated in FIGS. 1-8 and FIG. 13. Thedocking station 220 comprises a first connector 222 and a secondconnector 224 for operational connection with the particlecharacterization means when the cartridge is received in the dockingstation. The docking station 220 comprises a first port 226communicating with the first port in the cartridge 210 for forming asubstantially tight gas connection between the first ports when thecartridge is inserted in the docking station. The substantially tightgas connection provides for application of a pressure causing a liquidflow through the first cavity and the second cavity.

The docking station 220 further comprises a second port 228communicating with the second port in the cartridge 210 for forming asubstantially tight gas connection between the second ports when thecartridge is inserted in the docking station. The substantially tightgas connection provides for application of a pressure causing a liquidflow through the opening.

The docking station 220 comprises a pump device 230 comprising one ormore pumps and one or more directional valves for application of apressure on the first port 226 and/or the second port 228. Further, thedocking station may comprise one or more engagement members (not shown)for engagement with and moving the first sampling member and/or thesecond sampling member when the cartridge is removably inserted into thedocking station. Operation of the pump device and the one or moreengagement members is controlled according to a desired method ofmeasuring.

Different features of the illustrated embodiments may be combined.

FIG. 15 illustrates a preferred embodiment of the method according tothe invention. In the illustrated embodiment, step a) comprises enteringa first and a second liquid sample containing particles into a first andsecond cavity, respectively. Step b) comprises moving a first liquidthrough the first cavity and into a mixing chamber together with thefirst liquid sample, and step c) comprises performing first particlecharacterizing measurements by passage of at least a part of the firstliquid sample from the mixing chamber through an opening and into acollection chamber. Step d) comprises moving a second liquid through thesecond cavity and into the mixing chamber together with the secondliquid sample, and step e) comprises performing second particlecharacterizing measurements by passage of at least a part of the secondliquid from the mixing chamber through the opening and into thecollection chamber. Preferably, steps b) and d) are performed asdescribed in connection with FIGS. 1 and 2.

The invention claimed is:
 1. A method for characterizing particles inliquid, comprising the steps of: a) entering first and second liquidsamples containing particles for simultaneous holding in respectivefirst and second cavities, b) moving first liquid through the firstcavity and into a mixing chamber her with the first liquid sample, c)performing first particle characterizing measurements by passage of atleast a part of the first liquid sample from the mixing chamber throughan opening and into a collection chamber, d) moving second liquidthrough the second cavity and into the mixing chamber together with thesecond liquid sample, and e) performing second particle characterizingmeasurements by passage of at least a part of the second liquid samplefrom the mixing chamber through the opening and into the collectionchamber, wherein the first and second cavities are disposed inrespective rotatable first and second sampling members.
 2. A methodaccording to claim 1, wherein step c) of performing first particlecharacterizing measurements comprises the step of counting of red bloodcells and platelets by Coulter Counting of at least a part of the firstliquid sample.
 3. A method according to claim 1, wherein step e) ofperforming second particle characterizing measurements comprises thestep of counting one or more different types of white blood cells in atleast a part of the second liquid sample.
 4. A method according to claim1, wherein step a) of entering first and second liquid samples comprisesmoving the first and second sample members to respective first positionsso that the first and second cavities are in flow communication witheach other.
 5. A method according to claim 4, wherein step b) of movingthe first liquid comprises moving the first sample member to a secondposition so that the first cavity is in flow communication with themixing chamber and a first storage chamber holding the first liquid. 6.A method according to claim 5, wherein step d) of moving the secondliquid comprises moving the second sample member to a second position sothat the second cavity is in flow communication with the mixing chamberand a second storage chamber holding the second liquid.
 7. A method forcharacterizing particles in liquid, comprising the steps of: a) enteringfirst and second liquid samples containing particles for simultaneousholding in respective first and second cavities, b) moving first liquidthrough the first cavity and into a mixing chamber together with thefirst liquid sample, c) performing first particle characterizingmeasurements by passage of at least a part of the first liquid samplefrom the mixing chamber through an opening and into a collectionchamber, d) moving second liquid through the second cavity and into themixing chamber together with the second liquid sample, and e) performingsecond particle characterizing measurements by passage of at least apart of the second liquid sample from the mixing chamber through theopening and into the collection chamber, wherein the first and secondcavities are disposed in a rotatable sampling member.
 8. A methodaccording to claim 7, wherein step a) of entering first and secondliquid samples comprises moving the sample member to a first position sothat the first and second cavities are in flow communication with eachother.
 9. A method according to claim 8, wherein step b) of moving thefirst liquid comprises moving the sample member to a second position sothat the first cavity is in flow communication with the mixing chamberand a first storage chamber holding the first liquid.
 10. A methodaccording to claim 9, wherein step d) of moving the second liquidcomprises moving the sample member to a third position so that thesecond cavity is in flow communication with the mixing chamber and asecond storage chamber holding the second liquid.
 11. A method forcharacterizing particles in liquid, comprising: entering first andsecond liquid samples containing particles respectively into first andsecond cavities; changing a position of the first cavity to move a firstliquid through the first cavity and into a mixing chamber together withthe first liquid sample; performing first particle characterizingmeasurements by passage of at least a part of the first liquid samplefrom the mixing chamber through an opening and into a collectionchamber; changing a position of the second cavity to move a secondliquid through the second cavity and into the mixing chamber togetherwith the second liquid sample; and performing second particlecharacterizing measurements by passage of at least a part of the secondliquid sample from the mixing chamber through the opening and into thecollection chamber, wherein the first and second cavities are disposedin respective rotatable first and second sampling members; and whereinsaid entering first and second liquid samples comprises moving the firstand second sample members to respective first positions so that thefirst and second cavities are in flow communication with each other. 12.A method according to claim 11, wherein said performing first particlecharacterizing measurements comprises counting red blood cells andplatelets of at least a part of the first liquid sample.
 13. A methodaccording to claim 11, wherein said performing second particlecharacterizing measurements comprises counting one or more differenttypes of white blood cells in at least a part of the second liquidsample.
 14. A method according to claim 11, wherein said changing aposition of the first cavity comprises moving the first sample member toa second position so that the first cavity is in flow communication withthe mixing chamber and a first storage chamber holding the first liquid.15. A method according to claim 14, wherein said changing a position ofthe second cavity comprises moving the second sample member to a secondposition so that the second cavity is in flow communication with themixing chamber and a second storage chamber holding the second liquid.16. A method for characterizing particles in liquid, comprising:entering first and second liquid samples containing particlesrespectively into first and second cavities; changing a position of thefirst cavity to move a first liquid through the first cavity and into amixing chamber together with the first liquid sample; performing firstparticle characterizing measurements by passage of at least a part ofthe first liquid sample from the mixing chamber through an opening andinto a collection chamber; changing a position of the second cavity tomove a second liquid through the second cavity and into the mixingchamber together with the second liquid sample; and performing secondparticle characterizing measurements by passage of at least a part ofthe second liquid sample from the mixing chamber through the opening andinto the collection chamber, wherein the first and second cavities aredisposed in a rotatable sampling member, and wherein said entering firstand second liquid samples comprises moving the sample member to a firstposition so that the first and second cavities are in flow communicationwith each other.
 17. A method according to claim 16, wherein saidchanging a position of the first cavity comprises moving the samplemember to a second position so that the first cavity is in flowcommunication with the mixing chamber and a first storage chamberholding the first liquid.
 18. A method according to claim 17, whereinsaid changing a position of the second cavity comprises moving thesample member to a third position so that the second cavity is in flowcommunication with the mixing chamber and a second storage chamberholding the second liquid.